人乳头瘤病毒6型L1蛋白的原核表达及免疫原性评价

Prokaryotic expression and immunogenicity evaluation of human papillomavirus type 6 L1 protein

目的确定原核表达系统中人类乳头瘤病毒6型(human papillomavirus 6,HPV6)L1蛋白的最佳表达条件,并对HPV6 L1 VLP免疫小鼠后产生的中和抗体水平进行评价。方法将密码子优化的HPV6 L1基因片段与原核表达载体pET30a(+)连接,构建重组表达质粒pET30a-6L1,在大肠埃希菌中表达HPV6 L1蛋白,并通过添加分子伴侣(TF、GroES-GroEL、DnaK-DnaJ-GrpE)筛选能够增加目的蛋白表达的最佳条件。将表达的HPV6 L1蛋白经密度梯度离心、阳离子交换层析后获得纯化蛋白,对其形态进行表征。将纯化的HPV6 L1 VLP免疫BALB/c小鼠,采用假病毒中和试验检测小鼠血清中和抗体水平。结果质粒pET30a-6L1经双酶切鉴定证明构建正确;添加TF分子伴侣时HPV6 L1蛋白表达量最高;HPV6 L1 VLP是呈均一的、直径45~65 nm的球状结构,与HPV6天然病毒颗粒的形态、大小相似;HPV6 L1 VLP初次免疫小鼠第8周,血清抗体水平达到峰值,为105以上。结论分子伴侣TF促进了HPV6 L1蛋白的可溶性表达,纯化后的目的蛋白免疫原性良好。本研究为后续各型别HPV L1蛋白表达工艺的探索提供了思路,也为下一步的中试扩大培养奠定了基础。

Objective To optimize the expression condition of L1 protein of human papillomavirus 6(HPV6)in prokaryotic expression system,and evaluate the neutralizing antibody level in mice immunized with HPV6 L1 VLPs.Methods The HPV6 L1 gene fragment with optimized codon was linked to prokaryotic expression vector pET30a(+)to construct recombinant expression plasmid pET30a-6L1 for expression of HPV6 L1 protein in E.coli,and the expression condition for increasing the expression of target protein were optimized by adding molecular chaperones(TF,GroESGroEL and DnaK-DnaJ-GrpE).The expressed HPV6 L1 protein was purified by density gradient centrifugation and cation exchange chromatography,of which the morphology was characterized.BALB/c mice were immunized with the purified HPV6 L1 VLPs,of which the serum neutralizing antibody level was determined by pseudovirus neutralization test.Results Restriction analysis proved that recombinant plasmid pET30a-6L1 was constructed correctly.The expression level of HPV6 L1 protein was the highest when TF chaperone was added.HPV6 L1 VLPs were uniform spheres at diameters of 45~65 nm,of which the morph ology and size were similar to those of natural HPV6 particles.The serum antibody level of mice reached the peak value of more than 105 at the 8th week after the first immunization with HPV6 L1 VLP.Conclusion Molecular chaperone TF promoted the soluble expression of HPV6 L1 protein,and the purified target protein showed good immunogenicity,which provided references for investigation on the expression process of HPV L1 protein of various types,and also laid a foundation of further pilot culture.